1. Field of the Invention
The present invention relates to peptidyl prolyl-cis, trans isomerases (PPIase) derived from Escherichia coli and yeast, methods of producing the same, and genes coding for the same.
The PPIase accelerates a protein folding reaction by catalyzing the isomerization of prolyl peptide bonds in the protein. It is useful as a means for activating an inactive protein produced by genetic engineering and as a reagent for enzymological analysis. Its corresponding gene coding is useful for the production of the enzyme by genetic engineering, and it is also expected to be used as starting material for the gene which allows for the production, by genetic engineering, of the enzyme derivatives in which the enzymatic activity, intracellular localizability, substrate specificity and stability thereof have been altered. The present invention further provides means for efficient production of a useful protein having a correct conformation by expressing the above-mentioned gene in the same cells [as that] in which other gene coding for the useful protein is expressed.
2. Description of the Related Art
PPIase was found in porcine kidneys (Fisher, G., Bang, H. & Mech, C. Biomed. Biochim. Acta, 43, 1101-1111 (1984)], and is known to have the enzymatic activity of isomerizing an X.sub.aa -Pro bond (wherein X.sub.aa stands for any amino acid and Pro for L-proline) [Fisher, G., Bang, H. & Mech, C., Biomed. Biochim. Acta, 43, 1101-1111 (1984)] and to accelerate a protein folding reaction in some denaturated proteins such as immunoglobulin L chain and ribonuclease T.sub.1 [Lang, K., Schmid, F. & Fisher, G., Nature, 329, 263-210 (1987)]. The amino acid sequence of the enzyme purified from porcine kidneys was shown to be identical with that of cyclophilin, a protein known to bind to an immunosuppressive agent, cyclosporine A (CsA) which has been found to inhibit the PPIase activity. Since the immunosuppressive activity is directly proportional to the binding ability of CsA derivatives to cyclophilin, which represents most of the CsA-binding activity in lymphoid cells, it is inferred that the action of CsA, for example, an immunosuppressing action in T-cells is mediated through the inhibition of the PPIase activity [Takanashi, N., Hayano, E. & Suzuki, M., Nature, 337, 473-475 (1989)]. The CsA binding and the PPIase activities were found in almost all organs and an nearly all species. However it is not known how CsA acts specifically on the immune system and particularly on T cells, during an organ transplantation, this action has not been explained.
Rhodopsin, a visual pigment occurring in animal retinas, consists of a chromophore, 11-cis-retinal, bonded to opsin in the protein portion of rhodopsin. It is associated with visual transduction in the photoreceptor cells in which the chromophore is gradually converted and the maximum wavelength of absorption is consequently varied under the influence of light. In Drosophila a mutant in which the conversion of the precursor opsin to the rhodopsin is inhibited, is known. The ninaA gene responsible for this inhibition has been isolated and a nucleotide sequence thereof has been determined. As a result, it has been demonstrated that the ninaA gene codes for a ptotein having a homology to cyclophilin in the amino acid sequence. Accordingly, since the ninaA codes for a cyclophilin-like protein and the cyclophilin is identical with the PPIase, the ninaA gene probably encodes a protein possessing a PPIase activity. Thus, its activity is assumed to effect the formation of rhodopsin from its precursor opsin by controlling its folding reactions. The ninaA gene is exclusively expressed in the head part containing photoreceptor cells, and in the other parts of the body, gene fragments hybridizing with the ninaA gene and possessing a different size therefrom are detected. Consequently, it is inferred that the ninaA gene is specifically expressed in the photoreceptor cells and functions only in the formation of rhodopsin [Shieh, B.-H., Stamnes, M. A, Seavello, S., Harris, G. L. & Zuker, C. S., Nature, 338, 67-70 (1989); and Schneuwly, S., Shortridge, R. D., Larrivce, D. C., Ono, T., Ozaki, M. & Pak, W. L., Proc. Natl. Acad. Sci USA., 86 (1989)].
Likewise, if a PPIase is specifically expressed in cells the presence of the T-cell specific form may offer an explanation for the specific effect of CsA on the T cells. This assumption is supported by the observation that many gene copies capable of hybridizing with the cyclophilin gene are present in mammalian cells [Haendler, B., Hofer Warbinek, R. & Hofer, E., EMBO J., 6, 547-950 (1987)]. Only one kind of cyclophilin is confirmed to be expressed in each cell to far analyzed, and no multiplicity is found in the protein so expressed. A proof of the presence of several cyclophilins or PPIases in one species may well be theoretically accepted as evidence that each PPIase has a specific protein substrate.
In the case of Neurostora the presence of two species of cyclophilin mRNA transcribed from a single gene is known. One of these mRNAs codes for the cyclophilin molecule present in the cytoplasm, and another mRNA codes for a mitochondrial form having an additional signal sequence for its translocation to mitochondria. The latter protein, after translocation in the mitochondrion, is processed and forms a molecular species identical with the cyclophilin present is the cytoplasm. In this case, therefore, one molecular species of active protein is actually present and the multiplicity is ascribable to a difference in the localization of one protein in the cell [Tropschug, M., Nicholson, D. W., Hartl, F.-U., Kohler, H., Pfanner, N., Wachter, E. & Neupert, W., J. Biol. Chem. 263, 14433-14440 (1988)].
The catalytic effect of PPIase on the protein folding was investigated in nine kinds of proteins, i.e., concanavalin A, prothrombin, ribonuclease A, ribonuclease T.sub.1, cytochrome C, .beta.-lactoglobulin, meiokinase, chymotrypsinogen, and pepsinogen, which are known to be such that, during the refolding of the protein from the denatured state to the active state, the isomerization of the prolyl-containing peptide bonds constitutes the rate-determining step in the reconstruction of the denatured protein. However, the refolding of only two kinds of these proteins, i.e., ribonuclease T and cytochrome C were found to be accelerated by PPIase [Lin, L-N., Hasumi, H. & Brands, J. F., Biochim. Botphys. Acta 956, 256-266 (1988)]. These results suggest that one species of PPIase can act upon limited species of protein substrates.
Although, based on the facts described above, the presence of multiple forms of PPIase acting on restricted kinds of proteins is presumed, the presence thereof has not been actually proved.
The theory that multiple forms of PPIase are present in one species of organism, and that the substrate specificity of a given PPIase, for example, from a mammal, which is constituted from a large number of cells endowed with highly differentiated functions, may differ from that, for example, of yeast which is a unicellular organism and Escherichia coli which is a prokaryote, is believed to be a logical conclusion in view of the different functions to be fulfilled by the different cells mentioned above.
The hypothesis that the action of CsA is modified through the inhibition of the PPIase activity has been proposed on the basis of the inhibitory effect of CsA on the porcine PPIase. To justify the hypotesis, it is important to clarify the question whether or not the activities of PPIases from many organisms of widely diverging phylogenic origins, for example, Escherichia coli and yeast, are inhibited by CsA. Moreover, it is not correlated yet between the distribution of the CsA-binding activity in lower organism and the inhibitory effect of CsA on their PPIases.
The presence of multiple forms of PPIases in various cells suggest that each PPIase acts on its specific substrates in each cell. This point, coupled with the finding that a cell specific form of PPIase may be present in the photoreceptor cells of Drosophila evidently acquires a profound significance in the use of a recombinant DNA for the production of a specific protein with a specific cell as a host. It is desirable that PPIases which affect the folding of the targeted protein or the process of the protein synthesis thereof, coexist with the targeted protein produced in the host cell. Generally, it is considered that the PPIase of the host cell effectively acts on the protein inherent in the host cell. Therefore, the PPIases and their genes of the Escherichia coli and yeast, which are frequently used as hosts for the production of useful substances by the recombinant DNA technology, may be useful for the purpose mentioned above. Various organisms are being studied for the presence of cyclophylin, using as an index the protein's ability to bond cyclosporin A, and the cyclosporin A bonding activity has been detected in arthropoda (cockroaches), trematoda, mollusks, molds, porifera, yeasts, and plants (pumpkins) as well as in mammals [Koletsky, A. J., Harding, M. W. & Handschumacher, R. E., J. Biol. Chem., 137, 1054-1059, 1986]. Nevertheless, no correspondence has been established between these activities and the PPIase activity.
Though it has been demonstrated that the porcine PPIase accelerates the folding of a protein, the question of whether or not the PPIase is actually present and exhibits the activity in the species, such as yeast and microbacteria, etc., and the question of the extent to which it participates in the folding of protein, has not been definitely answered. As one of the means for solving the numerous problems mentioned above, it is believed important to isolate PPIases from Escherichia coli and yeast, to study the nature of the PPIases as protein, and obtain the genes thereof.